Phosphorylation and dephosphorylation are metabolically significant reactions in physiological systems. These reactions occur at hydroxyl groups of certain proteins, at nitrogen atoms of certain other proteins, and at carboxyl groups of still more proteins, in each case having an effect on the activity and function of these proteins in vivo. Certain phosphorylation/-dephosphorylation reactions are mediated by protein kinases and phosphatases and their occurrences provide an indication of the activity of these enzymes. Abnormalities in phosphorylation/dephosphorylation reactions have been implicated in cell carcinogenesis, in allergic disorders, and in Alzheimer's disease. Protein phosphorylation is an essential process in all living cells, for signal transduction, apoptosis, proliferation, differentiation, and metabolism. Methods for the determination of the phosphorylation status of proteins are thus valuable in exploring the molecular origins of diseases and in the design of new drugs.
Certain metalloenzymes that contain two metal ions are known to catalyze dephosphorylation reactions, in particular the hydrolysis of phosphate diesters. Studies of the mechanism by which these reactions occur have involved the use of synthetic organic ligands that form dimetallic complexes and perform the same catalytic function. Most of these synthetic ligands were found to have low solubility in water, however, which limited their utility in laboratory studies. To remove this obstacle, a water-soluble ligand, 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane, has been developed. Disclosures of Zn(II) complexes of this ligand and its use in the hydrolysis of RNA are published by Iranzo, O., et al., “Cooperativity between Metal Ions in the Cleavage of Phosphate Diesters and RNA by Dinuclear Zn(II) Catalysts,” Inorganic Chemistry 42(24), 7737-7746 (2003), and Iranzo, O., et al., “Physical and Kinetic Analysis of the Cooperative Role of Metal Ions in Catalysis of Phosphodiester Cleavage by a Dinuclear Zn(II) Complex,” J. Am. Chem. Soc. 125(7), 1988-1993 (2003). Further studies of this molecule and of the transition state that it forms with the phosphate esters are reported by Yang, M.-Y., et al., “Substrate specificity for catalysis of phosphodiester cleavage by a dinuclear Zn(II) complex,” Chem. Commun. 2003, 2832-2833, and Yang, M.-Y., et al., “A transition state analog for phosphate diester cleavage catalyzed by a small enzyme-like metal ion complex,” Bioorganic Chemistry 35, 366-374 (2007).